Soybeans are a major food source for the United States, supplying 70-80% of its vegetable oil and over $350 million/year of edible protein products. Although most of this product exists as soy flour, about 12% of its total volume, and 50% of its value, lies in the soy protein isolate.
A typical process for isolating soy protein is shown in FIG. 1. Before entering the three-stage extractor, the raw soybeans are cracked, dehulled, and extracted with hexane to remove the soybean oil from the resulting soy flakes. The flakes are then desolventized and extracted in three stages with water at about pH 8.5 to disperse the protein and other soluble constituents present in the flakes. A centrifuge (C.sub.1) separates spent flakes from the protein-rich mother liquor. The mother liquor contains water dispersible proteins, some of which are acid-precipitable (aglobulins), those which are acid soluble (albumins), oligosaccharides (sucrose, stachyose, raffinose, and the like), salts, phytin, and other constituents. The protein is subsequently precipitated from the mother liquor by adding a suitable acid such as HCl to adjust the pH to about 4.5, the approximate isoelectric point of the major protein components, to produce a protein curd. While removing much of the soy protein from the mother liquor, this treatment does not precipitate acid-soluble proteins such as albumins. A second centrifugation (C.sub.2) separates the acidic liquor or "whey" from the protein-rich precipitate or "curd". The curd is then washed with water to remove any additional acidic whey, along with other soluble components in the whey, and is separated from the whey by a third centrifugation (C.sub.3). The curd is redispersed by adjusting the pH to about 7 with suitable alkalies. The resulting dispersion, usually about 13-18% weight protein, is spray-dried to yield the soy protein isolate as a proteinate.
As a rough example based on using the equivalent of about 100 kg of moisture-free flakes, the existing soy protein isolation process produces about 33 kg of water insoluble spent flakes, 33 kg of dried protein isolate, and 33 kg of dissolved solids in the whey. The unprecipitated proteins present in the whey represent about 10% of the dispersible proteins present in the mother liquor. This failure to recover a substantial percentage of the protein in the soybeans reduces the utility of the existing process. More importantly, the loss of these proteins can compromise the functional and nutritional quality of the final product by affecting the overall protein composition. Additionally, these proteins contain a significant amount of phytin.
The flakes used for protein production are not sterile, and if produced under the best available conditions may have plate counts of under 25,000 microorganisms per gram of flakes. It is desirable to use flakes with as low a plate count as possible. All flakes must be essentially free of possible pathogens and common enteric organisms.
In the current processes, the extraction of the defatted flakes or flour to obtain the mother liquor is usually carried out at temperatures in the range of about 80.degree. F. to 150.degree. F. (27.degree. to 66.degree. C.). Further processing to separate the spent flakes from the mother liquor by centrifugation, precipitation of the curd, washing the curd and centrifugation to separate the curd from the whey and wash water, curd neutralization and other possible operations are carried out at temperatures of 80.degree. F. to 120.degree. F. (27.degree. to 49.degree. C.). The time period for the flake extraction may be 30 to 40 minutes for the initial extraction and an equal time for the subsequent washing of the extracted flakes. The subsequent operations (centrifugation, precipitation, curd washing, neutralization, and so forth) may require an additional 3 to 4 hours or more before the concentrated protein materials are dried. During that period of time, there may be a substantial increase in microbial population which can adversely affect the flavor, aroma, functionality, and color of the final product.
The temperatures used in the existing processing procedures are those which are generally ideal for the growth of many microorganisms. The protein solutions also contain ideal nutrients for microbial growth. When a given microbial population is exposed to conditions which promote growth, there is an initial "lag phase" in which there is little or no growth, after which the organisms begin to reproduce at an ever-increasing rate. Since the rate of growth of microorganisms is such that in short periods of time there is essentially a doubling of the microbial population, bacterial problems may result at the completion of processing even though only a small increase in microbial population was observed during the first hour or so. With dry products at low moisture levels below about 155 ppm, even though viable organisms may be present, they will not reproduce.
Assuming the bacterial count of the flakes is 1000/g, and assuming 8 parts of water per part of flake are used to make a slurry in order to prepare the mother liquor, the fresh slurry would have a count of about 125 organisms/ml. If it is assumed that the lag time is 15 minutes, after which the bacterial population doubles every 15 minutes, then after 15 minutes of extraction, the bacterial counts would be 125/ml; after 30 minutes, 250/ml; after 1 hour, 1000/ml; after 2 hours, 16,000/ml; after 3 hours, 256,000/ml; after 4 hours, 4,000,000/ml; after 5 hours, 66,000,000/ml; and so on. Depending on the types of organisms present and specific conditions of nutrient concentration, temperature, pH, and the like, the lag time and regeneration time could be somewhat less or considerably higher than in this example. Bacterial counts in the range of 1000-5000/ml would not be considered high, but counts in the several hundred thousand to the millions level per ml are undesirable.
Crosslinked poly(N-isopropylacrylamide) gels have been prepared which exhibit strongly temperature-dependent swelling when exposed to water. At low temperatures, these gels are in an expanded phase, and retain large amounts of water, along with any low molecular weight solutes which may be dissolved in the water. At only slightly higher temperatures, the gels undergo a phase transition whereby they contract in volume and exclude water and low molecular weight solutes, preferring interactions between the polymer molecules. These phase transitions involve an unusual form of critical point, a lower critical solution temperature (LCST).
The large changes in volume of crosslinked poly(N-isopropylacrylamide) gels which occur when the gels are heated or cooled through narrow temperature ranges can be analogized to the phase transition behavior of a van der Waals fluid, as shown in FIG. 2. FIG. 2(a) is a pressure-volume graph for a van der Waals fluid. Starting in the gas region at low pressures and raising the pressure at a constant temperature along the isotherm shown, the two-phase envelope is eventually reached at point A. The pressure corresponding to point A is the equilibrium vapor pressure at which the gas begins to condense and at which both gas and liquid coexist as separate phases. The fluid volume drops sharply as vapor condenses to a liquid.
The dotted horizontal line containing point A is the two-phase segment of but one of many isotherms that can exist for a van der Waals fluid. Other horizontal segments can be drawn at pressures between that of point A and the critical pressure P.sub.c which corresponds to the pressure at the top of the two-phase envelope. This critical pressure is the greatest pressure at which both liquid and vapor can coexist. The critical pressure P.sub.c also corresponds to a critical temperature, T.sub.c, the isotherm of which passes through the point at the top of the two-phase envelope. At temperatures above T.sub.c, only the gas phase exists, no matter how great the pressure applied.
FIG. 2(b) shows the volume of a typical crosslinked poly(N-isopropylacrylamide) gel plotted versus temperature instead of pressure. The van der Waals gas phase can be thought of as corresponding to a "gel gas" phase, and the van der Waals liquid phase to a "gel liquid" phase. The "gel liquid" phase corresponds to the poly(N-isopropylacrylamide) gel in its "collapsed" state, while the "gel gas" phase represents the poly(N-isopropylacrylamide) gel in its "swollen" state. Thus, when processing temperatures are near the gel's critical temperature, i.e., its LCST, the collapsing and swelling of the gel can occur within very narrow temperature ranges and can be discontinuous.
The dramatic effect of temperature on the expansion and contraction of crosslinked N-substituted polyacrylamide gels was carefully studied by R. Freitas and E. Cussler, Chem. Eng. Science, 42, 97 (1987). The authors reported that crosslinked poly(N-isopropylacrylamide) gels and crosslinked copolymers of 97% N,N-diethylacrylamide and 3% sodium methacrylate which are swollen to equilibrium in water at 25.degree. C. collapse abruptly when heated to 33.degree. C. and to near 55.degree. C., respectively. The polymer has a natural limit as to water uptake, leaving excess water and water soluble materials as free water or in solution. In actual use, excess water should be present in order to obtain a retentate concentrated in protein (or whatever high molecular weight entities one may wish to concentrate), and of relatively low concentration of small molecular weight moieties.
These temperature sensitive gels have been used to concentrate macromolecular solutions, including those of proteins. In general, solutes with molecular weights above 10.sup.4 daltons are completely excluded by the gels, and solutes with molecular weights below 10.sup.3 daltons diffuse freely into the gels. (See S. H. Gehrke et al., Chem. Eng. Science, 41, 2153 (1987)). However, the protein concentration experiments published to date have been in highly idealized model systems, often containing only one protein or a few low molecular weight solutes, all at high dilution. The few published experiments made under relatively high concentrations show poor separation. Moreover, changes in pH and ionic strength can cause different, sometimes unexpected phase transitions. For example, when using poly(N-isopropylacrylamide) gel to concentrate gelatin from solution, the average efficiency (efficiency being defined as concentration increase measured in solution divided by that expected from the altered solution volume) observed for 0.5 wt-% solutions of gelatin was above 95%. However, for a solution containing 5 wt-% gelatin, the separation efficiency was only 60%. The reduced separation efficiency was attributed to small amounts of solution or "raffinate" becoming trapped between the particles of swollen gel or adsorbed onto the gel particle surfaces. In the existing processes using acid precipitation, the protein concentration in the clarified mother liquor can be in the range of 5 wt-%, and the concentration of non-protein solubles is about 3 wt.-%. Following the acid precipitation step, less than about 90% of the total protein in the mother liquor is recovered as the curd.
Therefore, there is a need for a soy protein isolation process which not only concentrates the acid-soluble proteins, presently not recovered by the acid-based method, but also which achieves a greater separation efficiency for feed solutions more highly concentrated in protein. Such an isolation process would result in improved economics, alleviate waste disposal problems, and provide products with improved functional characteristics and nutritional value.